The present invention relates to solid phytase compositions which have been stabilized with a lactic acid source such as Corn Steep Liquor (CSL), and methods of producing the same.
The addition of phytase to animal feed to eliminate the anti-nutritional effects of phytic acid is well-described, see e.g. WO 98/28408 and WO 98/28409.
The stabilization of liquid phytase formulations with urea, glycerol or sorbitol is disclosed in WO 93/16175.
Salt-stabilized solid phytase compositions are disclosed in EP 0 758 018 A1.
Plant seeds, cereal grains and legumes are usual components of animal feed. Some of those seeds contain phytic acid, and often also endogenous phytase enzymes.
According to investigations performed by the applicant, endogenous phytase activity in animal feed is at a very low level of around 0.5 units/g.
According to e.g. the two above first-cited WO-references, when supplementary phytase has been added to feed, the phytase activity in the feed is in the range of 0.01-20 units/g.
The present invention relates to solid phytase compositions which comprise (a) an enzyme having phytase activity; and (b) a lactic acid source, wherein the phytase activity of the composition is above 20 units/g.
In the present context, the expression xe2x80x9cenzyme (or polypeptide) having phytase activityxe2x80x9d or xe2x80x9cphytasexe2x80x9d includes any enzyme capable of effecting the liberation of inorganic phosphate from phytic acid or from any salt thereof (phytates).
Phytic acid is myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate (or for short myo-inositol hexakisphosphate). In what follows, unless otherwise indicated, the terms xe2x80x9cphytic acidxe2x80x9d and xe2x80x9cphytate,xe2x80x9d are used synonymously or at random.
In the present context, the term xe2x80x9cunitsxe2x80x9d means units of enzyme, in particular phytase, activity. Any method for determining phytase activity can be used.
In a preferred embodiment, one unit of phytase activity is defined as the amount of enzyme that liberates 1 micro mole inorganic ortho-phosphate per min. under the following conditions: A pH which is within the range of +/xe2x88x921 pH unit from the optimum pH of the actual enzyme; a temperature which is within the range of +/xe2x88x9220xc2x0 C. from the optimum temperature of the actual enzyme; using as a substrate phytic acid or any salt thereof in a suitable concentration.
Preferably, the substrate is dodeca-sodium phytate in a concentration of 0.005 mole/l.
Preferably, the pH is within the range of +/xe2x88x920.5 pH unit from the optimum pH; more preferably the pH is the optimum pH.
Preferably, the temperature is within the range of +/xe2x88x9210xc2x0 C. from the optimum temperature; more preferably the temperature is the optimum temperature.
Preferably, the optimum pH and optimum temperature refers to the use of sodium phytate as a substrate.
In another preferred embodiment, the phytase activity is determined in the unit of FYT, one FYT being the amount of enzyme that liberates 1 micro mole inorganic ortho-phosphate per min. under the following conditions: pH 5.5; temperature 37xc2x0 C.; substrate: sodium phytate (C6H6O24P6Na12)in a concentration of 0.0050 mole/l.
In a further preferred embodiment, the phytase activity is measured using the FTU assay.
The FYT- and FTU-assays are described in more detail in the experimental part.
In preferred embodiments, the phytase activity of the solid composition of the invention is above 25, 50, 100, 250, 500, 750 or even above 1000 units/g.
Optionally, the phytase activity of the solid composition is below 100,000 units/g, more preferably below 75,000 units/g, even more preferably below 50,000 units/g, or below 40,000 units/g, or below 25,000 units/g, or even below 10,000 units/g, mostly preferred below 5,000 units/g.
Preferred ranges of phytase activity are 25-100,000, 25-75,000, 35-50,000, or 50-40,000 units/g; more preferably 100-25.000 units/g; even more preferably 500-10.000 units/g; mostly preferred 1000-5000 units/g.
In the present context, any enzyme having phytase activity can be used.
Phytases have been derived from plants as well as from microorganisms. Amongst the microorganisms, phytase producing bacteria as well as phytase producing fungi are known. From the plant kingdom, e.g. a wheat-bran phytase is known (Thomlinson et al, Biochemistry, 1 (1962), 166-171). An alkaline phytase from lilly pollen has been described by Barrientos et al, Plant. Physiol., 106 (1994), 1489-1495.
Amongst the bacteria, phytases have been described which are derived from Bacillus subtilis (Paver and Jagannathan, 1982, Journal of Bacteriology 151:1102-1108) and Pseudomonas (Cosgrove, 1970, Australian Journal of Biological Sciences 23:1207-1220). Still further, a phytase from E. coli has been purified and characterized by Greiner et al, Arch. Biochem. Biophys., 303, 107-113, 1993).
Phytase producing yeasts are also described, such as Saccharomyces cerevisiae (Nayini et al, 1984, Lebensmittel Wissenschaft und Technologie 17:24-26. However, this enzyme is probably a myo-inositol monophosphatase (Wodzinski et al, Adv. Appl. Microbiol., 42, 263-303). AU-A-24840/95 describes the cloning and expression of a phytase of the yeast Schwanniomyces occidentalis. 
There are several descriptions of phytase producing filamentous fungi, primarily belonging to the fungal phylum of Ascomycota (ascomycetes). In particular, there are several references to phytase producing ascomycetes of the Aspergillus genus such as Aspergillus terreus (Yamada et al., 1986, Agric. Biol. Chem. 322:1275-1282). Also, the cloning and expression of the phytase gene from Aspergillus niger var. awamori has been described (Piddington et al., 1993, Gene 133:55-62). EP 0 420 358 describes the cloning and expression of a phytase of Aspergillus ficuum (niger). EP 0 684 313 describes the cloning and expression of phytases of the ascomycetes Myceliophthora thermophila and Aspergillus terreus. 
Phytases derived from fungi of the phylum Basidiomycota are disclosed in WO 98/28409 and WO 98/28408.
Modified phytases or phytase variants are obtainable by methods known in the art, in particular by the methods disclosed in EP 0897010, EP 0897985, PCT/DK99/00153 and PCT/DK99/00154. The phytases disclosed in either of these four patent applications can also be used in the compositions of the present invention.
A solid or dry composition is a particulate material comprising, preferably consisting essentially of, or consisting of, freely flowing particles of a size ranging from (xcexcm) 0.01, or from 1.0, or preferably from around 1-to 1000, or to 1200, or to 1500, or even up to 2000 (xcexcm).
Preferably, a solid or dry phytase composition is such composition which can be prepared from liquid phytase concentrates e.g by spray drying, spray cooling (prilling), or any type of granulation.
For spray drying, no further components need to be added to the liquid phytase concentrate.
For spray cooling, a meltable componentxe2x80x94such as palm oil (and/or another meltable vegetable oil or fat), hydrogenated palm oil (and/or another hydrogenated vegetable oil), tallow, hydrogenated tallow or a wax functions as a matrix. The phytase and other ingredients, if any, are introduced into the melted, meltable component, and the melt is then allowed to solidify under particle-forming conditions, typically in a spray drying tower.
For many uses, however, including the use in animal feed, granulates are usually preferred for a number of reasons. One reason being that they may readily be mixed with feed components, or more preferably, form a component of a pre-mix which contains other desired feed additives such as vitamins and minerals.
The particle size of the enzyme granulates preferably is compatible with that of the other components of the mixture. This provides a safe and convenient mean of incorporating enzymes into, e.g., animal feed.
The size of a particle may be regarded as the greatest linear dimension of the particle; thus, in the case of, e.g., a substantially spherical particle (such as a substantially spherical granulate particle), the particle size in question will be the diameter of the particle.
Agglomeration granulates and agglomerated powders may be prepared using agglomeration technique in a high shear mixer (e.g. Lxc3x6dige) during which one or more filler materials and the enzyme are co-agglomerated to form granules.
Absorption granulates are prepared by having cores of one or more carrier materials to absorb/be coated by the enzyme.
Typical filler materials are salts such as di-sodium sulphate and calcium-lignosulphonate. Other fillers are silica, gypsum, kaolin, talc, magnesium aluminium silicate and cellulose fibres. Optionally, binders such as dextrins are also included in agglomeration granulates.
Typical carrier materials may consist of particulate cores having a suitable particle size. The carrier may be water soluble or water insoluble, e.g. starch, e.g. in the form of cassava or wheat; or a sugar (such as sucrose or lactose), or a salt (such as sodium chloride or sodium sulphate).
Optionally, the granulates are coated with a coating mixture. Such mixture comprises coating agents, preferably hydrophobic coating agents, such as hydrogenated palm oil and beef tallow, and if desired other additives, such as calcium carbonate or kaolin.
WO 97/39116 discloses preferred processes for making solid compositions of the invention in the form of enzyme-containing granules or an enzyme-containing granulate, see in particular the sections of the detailed description therein headed cores, binders, fillers, plasticizers, fibrous materials, superabsorbents, coating layers, enzymes, other adjunct ingredients (these sections being hereby incorporated by reference herein). However, WO 97/39116 does not disclose the inclusion in the solid composition of a lactic acid source. Preferred methods of preparing phytase granulates are referred to in Example 3.
Preferred solid compositions of the invention are enzyme compositions. The preferred compositions are concentrated, viz. of an activity of above 20 units/g. Thus, the concept of solid enzyme composition comprises in particular, but are not limited to, spray-dried enzyme preparations, enzyme granulates, e.g. agglomeration granulates and absorption granulates, coated as well as un-coated, and enzyme-containing pre-mixes for animal feed. Phytase is a preferred enzyme.
Liquid enzyme (phytase) concentrates can e.g. be prepared as follows: The enzyme source, typically a phytase-containing fermentation broth, is subjected to a primary separation step (e.g. using a decanter, a centrifuge, or a filter press), followed by a second polish filtration and/or germ filtration step. Finally the liquid is concentrated, e.g. using ultra filtration, followed by a germ filtration. A typical dry matter content is in the range of 10-30%, preferably 15-25%, more preferably 17-22%.
In the present context, xe2x80x9caxe2x80x9d generally means xe2x80x9cone or morexe2x80x9d or xe2x80x9cat least one.xe2x80x9d This applies i.a. for the following mandatory or optional components of the compositions of the invention: Phytase, lactic acid source, CSL, starch, disaccharide, filler, carrier.
Unless otherwise indicated, all percentage indications are weight/weight, by reference to dry matter content. Preferably, xe2x80x9cunits/gxe2x80x9d also refers to dry matter content. Dry matter content can be determined by any method known in the art, such as refractometer or drying in an oven to release humidity.
Unless otherwise indicated, the expression xe2x80x9cabovexe2x80x9d generally means xe2x80x9cxe2x89xa7xe2x80x9d, whereas the expressions xe2x80x9cup toxe2x80x9d or xe2x80x9cbelowxe2x80x9d mean xe2x80x9cxe2x89xa6xe2x80x9d.
In the present context a xe2x80x9clactic acid sourcexe2x80x9d or a xe2x80x9clactic acid preparationxe2x80x9d is any composition which comprises the compound lactic acid or any lactates, i.e. any salts thereof (lactic acid is 2-hydroxy propanic acid). Likewise, xe2x80x9clactic acidxe2x80x9d as used herein includes any lactates. These expressions are used interchangeably for the lactic acid source, resp. the lactic acid, as is, and for the dry matter part thereof.
A non-limiting list of lactic acid sources is the following: Lactic acid and lactates as relatively pure chemical compounds (purity of, say, above 70%, 80%, 90%); lactic acid and lactates as more impure substances (purity of, say, above 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%); any natural or synthetical composition which comprises lactic acid in an amount of above 5%, preferably above 10%, 15%, 20%, 25%, 30%, 40%, 50%, 70%, 80%, 90%.
The solid enzyme compositions of the invention preferably comprise up to 20, preferably up to 15, more preferably up to 10, still more preferably up to 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75 or 0.5% lactic acid. The content of lactic acid is preferably above 0.001, preferably above 0.002, 0.004, 0.006, 0.008, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, 0.22, 0.24, 0.26, 0,28 or above 0.3%. Preferred ranges of content of lactic acid are 0.01-10%, 0.02-9%, 0.03-8%, 0.04-7%, 0.05-6%, 0.06-7% 0.07-6%, 0.08-5%, 0.09-4%, or 0.1-3%.
Any assay for lactic acid can be used. Preferred lactic acid assays are from SIGMA: (1) Assay kit catalogue no. 735-10 (enzymatical assay, lactate degraded to pyruvate and hydrogen peroxide in the presence of oxidase); or (2) Assay kit catalogue no. 826-A and 826-B (ultraviolet, endpoint, lactate converted into pyruvic acid in the presence of lactate dehydrogenase and NAD).
A preferred lactic acid source is Corn Steep Liquor or CSL. CSL is a commercially available product, see for instance Merck Index, 1996, 4th edition, Index no. 2598. It is a viscous yellowish or dense brown liquid obtained by concentration of corn steep water. The dry matter content is usually 45-55%, preferably 48-52%. The pH is in the range of 3-5, preferably 3.5-4.5. The protein content (Dry Matter) is typically 30-50%, preferably 35-45%. The acidity (as lactic acid) is typically 10-30% (Dry Matter), preferably 12-25%.
xe2x80x9cCSLxe2x80x9d as used herein refers to the product as such, or to its dry matter part.
In a preferred embodiment, the solid composition of the invention comprises 0.01-15%, preferably 0.1-10%, more preferably 1-5% of CSL.
For analyzing CSL-content, any method can be used. A preferred HPLC method for fingerprinting and quantifying CSL is indicated in Example 8. Another preferred method is Head Space Gas Chromatograph (HS-GC), preferably combined with mass spectrometry (MS).
In a preferred embodiment, the solid composition of the invention additionally comprises a starch source, typically in an amount of 0.1-20%, more preferably 0.2-10%, still more preferably 1-5%.
The concept of a starch source includes any natural or synthetic polysaccharides comprising glucose units interconnected by alpha-1,4- or alpha-1,6-linkages. Purity preferably above 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%. A preferred starch source is Wheat Starch, which is a commercially available product. The expression xe2x80x9cstarch sourcexe2x80x9d includes the starches and modified starches described in the section headed xe2x80x9cCoresxe2x80x9d of WO 97/39116, cited above.
In another preferred embodiment, the solid composition of the invention additionally comprises a disaccharide, preferably in an amount of 0.01-15%, even more preferred 0.1-10%, still more preferred 1-5%.
The concept of disaccharides includes any natural or synthetic disaccharides, whatever the monomers, and whatever the linkage type. Examples of such disacharides are maltose, lactose, cellobiose, sucrose, trehalose (non-limiting list). Preferably, the disaccharides are of a purity of above 10, 20, 30, 40, 50, 60, 70, 80 or even 90%. Preferred disaccharides are lactose and trehalose (alpha-D-glucose alpha-D-glucopyranoside, alpha-1,1 linkage).
In the process of the invention, all steps, e.g. those indicated in claim 14, can be performed simultaneously or sequenially. E.g. steps (i) and (ii) sequentially or preferably simultaneously (mixing the lactic acid source and the phytase before spraying it onto the carrier); steps (iii) and (iv) simultaneously or sequentially, preferably simultaneously, in the same apparatus; applies also to xe2x80x9ctogether withxe2x80x9d of claim 12.
Further preferred embodiments of the invention are the following:
A solid composition which comprises at least one enzyme having phytase activity, and Corn Steep Liquor (CSL), wherein the phytase activity of the composition is in the range of 20-50.000 units/g. A preferred amount of CSL is within the range of 0.01-15% (dry matter content and w/w). Preferably, the composition additionally comprises Wheat Starch (WS), preferably in an amount within the range of 0.01-20% (dry matter content and w/w);
A process for preparing a granulate composition having a phytase activity in the range of 20-50.000 units/g, which method comprises the steps of (i) spraying a liquid phytase concentrate onto a carrier; (ii) spraying CSL onto the carrier; (iii) mixing; and (iv) drying;
A process for preparing a spray dried solid composition having a phytase activity in the range of 20-50.000 units/g, which method comprises the step of adding CSL to a liquid phytase concentrate before spray drying it.
The activities of the solid phytase compositions prepared in the present examples are in the range of 1000-3000 FTU/g before storage.